Mechanical ventilation (MV) is used to sustain pulmonary gas exchange in patients incapable of maintaining adequate alveolar ventilation. The withdrawal of MV from patients is called "weaning" and problems in weaning are frequent. Studies reveal that diaphragmatic weakness is a key contributor to weaning difficulties. Importantly, we have discovered that MV-induced diaphragmatic weakness is associated with oxidative damage to the diaphragm. Moreover, our recent work reveals that MV-induced oxidative stress depresses diaphragmatic specific force production and promotes proteolysis leading to diaphragmatic atrophy. At present, the mechanism(s) responsible for MV-induced production of cellular oxidants are unknown. Therefore, these experiments will determine the oxidant pathway(s) responsible for MV-induced oxidative injury in the diaphragm. Based upon preliminary experiments, our working hypothesis is that MVinduced diaphragmatic oxidative stress occurs due to interactions between 4 oxidant-producing pathways: 1) production of superoxide radicals by NAD(P)H oxidase; 2) generation of superoxide radicals by the xanthine oxidase pathway; 3) nitric oxide production via nitric oxide synthase; and 4) formation of hydroxyl radicals by increased cellular levels of reactive iron. This hypothesis will be tested in a rat model of MV using an innovative and comprehensive experimental approach. First, we will measure MV-induced oxidative injury in the diaphragm using a panel of established techniques. Secondly, we will investigate critical elements of each oxidant production pathway to determine if these components are elevated in diaphragms from MV animals. Further, we will employ proven pharmacological inhibitors of each oxidant pathway to determine the relative importance of the pathway in MV-induced diaphragmatic oxidative injury and contractile dysfunction. Delineating the biochemical pathways responsible for MV-induced oxidant production in the diaphragm is essential to developing an effective approach to oppose this damaging process. These experiments will provide new and important mechanistic information about the source of oxidants in the diaphragm that can be used to develop optimal clinical strategies to retard MV-induced diaphragmatic oxidant stress and contractile dysfunction.